Adenylyl cyclases are a family of enzymes that catalyze the formation of cAMP from adenosine-5′-triphosphate (5′ATP), mediate the physiological effects of numerous hormones and neurotransmitters, and belong to a super family of membrane-bound transporters and channel proteins.
Adenosine-3′:5′-cyclic monophosphate (cAMP) is known to be the second messenger involved in signal transduction for numerous neurotransmitters and hormones, and thus has an impact upon some of the key mediators for SMC proliferation and migration. While it is known that the cAMP pathway can be regulated pharmacologically by inhibitory compounds that are of particular value in the treatment of many diseases, and there is still much interest in identifying more potent and specific agents acting on this pathway. Regulation of this pathway can be achieved through changes in the activities of cAMP-phosphodiesterases, cAMP-dependent protein kinases, or adenylyl cyclases.
Inhibitory compounds have been developed as therapeutic agents that inhibit cyclic nucleotide phosphodiesterases. Some effects of such agents are to raise cellular cAMP levels in tissues and organs on which they act. For example, theophylline, an inhibitor of all isozyme families of phosphodiesterases, is used clinically to treat asthma. Rolipram, an inhibitor of type IV phosphodiesterase, is used in the treatment of depression. And several inhibitors of type III phosphodiesterase have been used clinically to treat patients with moderate to severe heart failure. These latter drugs enhance cardiac index without elevating mean arterial blood pressure and lowering systemic vascular resistance. Therefore such compounds are believed to have significant advantages over .beta.-agonists and digitalis.
However, there is a continued need for discovering more effective and specific inhibitory compounds that act directly on adenylyl cyclases, even though inhibitory agents which indirectly activate or indirectly inhibit the enzyme may be commonly used in the treatment of such diseases. For example, drugs of the class beta-blockers are commonly used to treat hypertension and some of these act to inhibit adenylyl cyclase indirectly by blocking the stimulatory effects of the sympathetic nervous system to activate adenylyl cyclase in the heart, thereby reducing cardiac output. Agents that reduce adenylyl cyclase activity directly would be expected to have a similar cardiac-sparing effect, along with reduced cardiomyopathy and heart failure. Adenylyl cyclases can be potently and directly inhibited by analogues of adenosine, via a specific domain. This binding domain is referred to as the “P”-site from an evident requirement for an intact purine moiety. However, there is a need for more potent and direct adenylyl cyclase inhibitors.
Congestive heart failure (CHF) afflicts 3 to 4 million Americans and 400,500 to 500,000 new cases are diagnosed each year, and adenylyl cyclase plays a role in the disease progression, as discussed below. Significantly, statistics show that more than 50% of heart failure (CHF) patients die within five years of their diagnosis. It is believed to be the primary cause of 30,000 to 40,000 deaths annually.
CHF is defined as an abnormal heart function resulting in an inadequate cardiac output for metabolic needs. See E. Braunwald, Heart Disease, First Ed., W. B. Saunders, Philadelphia, page 426 (1988). The symptoms of CHF include for example, breathlessness, fatigue, weakness, leg swelling, and exercise intolerance. Initially, the conditions of patients with heart failure usually develop as the heart muscle weakens and needs to work harder to keep the blood flowing through the body. The heart failure develops, following an injury to the heart as damage caused by heart attack, long term high blood pressure or an abnormality of one of the heart valves. The weakened heart must then work harder to keep the demands of the body. Heart failure is usually not recognized until a more advanced stage of heart failure which is referred to as congestive heart failure. On physical examination, patients with CHF tend to have elevations in heart and respiratory rates, rates (an indication of fluid in the lungs), edema, jugular venous distension, and, in general, enlarged hearts. The most common cause of CHF is atherosclerosis which causes blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle. Ultimately, such blockages may cause myocardial infarction (death of heart muscle) with subsequent decline in heart function and resultant heart failure. Other causes of CHF include valvular heart disease, hypertension, viral infections of the heart, alcohol, and diabetes. Some cases of heart failure occur without clear etiology and are called idiopathic.
CHF is also typically accompanied by alterations in one or more aspects of β-adrenergic neurohumoral function; see Feldman et al., J. Clin. Invest., 82:189-197, (1988); Bristow et al., N. Engl. J. Med., 307:205-211, (1982); Bristow et al., Circ. Res., 59:297-309 (1986); Ungerer et al., Circulation, 87: 454-461 (1993); Bristow et al., J. Clin. Invest., 92: 2737-2745 (1993); Calderone et al., Circ. Res., 69:332-343 (1991); Marzo et al., Circ. Res., 69:1546-1556 (1991); C. S. Liang et al., J. Clin. Invest., 84: 1267-1275 (1989); Roth et al., J. Clin. Invest., 91: 939-949 (1993); Hadcock and Malbon, Proc. Natl. Acad. Sci., 85:5021-5025 (1988); Hadcock et al., J. Biol. Chem., 264: 19928-19933 (1989); Mahan, et al., Proc. Natl. Acad. Sci. USA, 82:129-133 (1985); Hammond et al., Circulation, 85:269-280 (1992); Neumann et al., Lancet, 2: 936-937 (1988); Urasawa et al., G Proteins: Signal Transduction and Disease, Academic Press, London. 44-85 (1992); Bohm, Mol. Cell Biochem., 147: 147-160 (1995); Eschenhage et al., Z. Kardiol, 81 (Suppl 4): 33-40 (1992); and Yamamoto et al., J. Mol. Cell., 26: 617-626 (1994). There are other references regarding various adenylyl cyclase enzymes. see, Yoshimura et al., Proc. Natl. Acad. Sci. USA, 89:6716-6720 (1992); Ishikawa et al., J. Biol. Chem., 267:13553-13557 (1992); Fujita et al., Circulation, 90(4): Part 2) (1994); Krupinski et al., J. Biol. Chem., 267:24858-24862 (1992); Ishikawa et al., J. Clin. Invest, 93:2224-2229 (1994); Katsushika et al., Proc. Natl. Acad. Sci. USA, 89:8774-8778 (1992); Wallach et al., FEBS Lett., 338:257-263 (1994); Watson et al., J. Biol. Chem., 269:28893-28898 (1994); Manolopoulos et al., Biochem. Biophys. Res. Commun., 208:323-331 (1995); Yu et al., FEBS Lett, 374:89-94 (1995); and Chen et al., J. Biol. Chem., 270:27525-27530 (1995).
Differential changes in the left and right ventricular adenylyl cyclase activities have been demonstrated in congestive heart failure patients (see, for example, Sethi, Rajat, et al., APStracts 3:0403H, 1196). The Sethi abstract reports that the levels of adenylyl cyclase in crude membranes from both left and right ventricles was determined upon occluding the left coronary artery in rats for 4, 8 and 16 weeks. The adenylyl cyclase activity in the presence of isoproterenol was decreased in the uninfarcted (viable) left ventricle and increased in the right ventricle subsequent to myocardial infarction. The catalytic activity of adenylyl cyclase was depressed in the viable left ventricle but was unchanged in the right ventricle. In comparison to sham controls, the basal, as well as NaF-, forskolin-, and Gpp(NH)p-stimulated adenylyl cyclase activities, were decreased in the left ventricle and increased in the right ventricle of the experimental animals. Opposite alterations in the adenylyl cyclase activities in left and right ventricles from infarcted animals were also seen when two types of purified sarcolemmal preparations were employed. These changes in adenylyl cyclase activities in the left and right ventricles were dependent upon the degree of heart failure. Furthermore, cyclic AMP contents were higher in the right ventricle and lower in the left ventricle from infarcted animals injected with saline, isoproterenol or forskolin in comparison to the controls. The results suggest differential changes in the viable left and right ventricles with respect to adenylyl cyclase activities during the development of congestive heart failure due to myocardial infarction. Accordingly, inhibiting adenylyl cyclase would be helpful in treating congestive heart failure.
Fibroproliferative vasculopathy includes restenosis following coronary bypass surgery and PTCA (percutaneous transluminal coronary angioplasty), allograft arteriosclerosis in chronic allograft rejection, diabetic angiopathy and all forms of common arteriosclerosis. Vascular intimal dysplasia and remodeling are characteristic features of reinjury following balloon angioplasty, coronary bypass surgery (Holmes et al. 1984; Holmes et al. 1988) and in chronic allograft rejection (Lemstrom and Koskinen, 1997; Hayry et al. 1993). An initial response to vascular injury is inflammatory and involves attraction of lymphocytes, macrophages and thrombocytes to the site of injury and secretion of cytokines, eicosanoids and growth factors (Ross 1993). Under the influence of growth factors and cytokines, smooth muscle cells (SMC) proliferate and migrate from the media to the intima and contribute to intimal hyperplasia and stenosis. Some key mediators of SMC proliferation and migration are IL-1, TNF alpha, PDGF, IGF1, bFGF, EGF, TGFβ and VEGF (Asahara et al. 1995; Bornfeldt et al. 1994; Ferns et al. 1991; Libby and Galis 1995, Galis et al. 1995; Gronwald et al. 1989; Hancock et al. 1994; Hayry et al. 1995; Lindner and Reidny 1991; Myllarnemi et al. 1997; Nabel et al. 1993; Shi et al. 1996; Tanaka et al. 1996) and the matrix metalloproteinases in SMC locomotion through the extracellular matrix (Bendeck et al. 1996; Galis et al. 1995).